Zoonotic Cutaneous Leishmaniasis (ZCL) is a disease highly prevalent in North Africa and Middle East that is caused by Leishmania major There is an urgent need for improved non toxic antileishmanial drugs and for effective vaccines that could prevent infection or symptomatic disease. The TMRC proposal aims at describing the natural history of L. major infection in Tunisia and identifying immune correlates of protection that takes into consideration the eventual effects of intraspecies polymorphism of L major and reservoir diversity The specific hypothesis is that genetic variation among L. major might act, in concert with the host immune response, to determine the outcome of infection. The specific aims of project 3 are to:1)Screen individuals with past history of ZCL, for the persistence of residual parasites in healed lesions.2) Establish a bank of L. major isolates from the ZCL lesions that will develop during the prospective follow up of a cohort of individuals exposed to the risk of infection. 3) Evaluate comparatively the intra-species functional diversity of these isolates using five experimental approaches: i- Kinetics of in vitro parasite growth in culture, ii- In vitro resistance to complement lysis, iii- In vitro infectivity to murine or human macrophages. iv- Experimental pathogenicity in inbred mouse strains, v- Quantification of the transcripts of a set ofpre- selected genes expressed in indicator human macrophages infected by the various isolates. 4) Select a subset of L. major isolates on the basis of the functional tests described above and analyze their proteomicand transcriptomic profiles to identify proteins differentially expressed among isolates. Data on L. major diversity generated by project 3 will be correlated to the clinical and immunological base line informations on individuals from whom the parasites were obtained (project 1 and project 2). Parasite strains with diverse functional phenotypes may help to identify virulence factors that might play key roles in host-parasite interactions and immune response . It will contribute to generate a holistic view on the natural history of ZCL considering the effect of the agent and to better understand the determinants of resistance to the disease.